The gene targeting projects that GEC members have worked on this year include the following scientific areas 1) Human disease modeling: It is often desirable to have mutations of human genetic conditions replicated in mouse so that the diseases can be modeled. In the last year, several such models have been made or are being developed in the NEI Genetic Engineering Core, in collaboration with NEI researchers. Mutations in the Fbn2 locus can lead to age-related macular degeneration (AMD) in human, which is often manifested in the elderly as gradual vision loss with age. One frequently encountered disease-generating mutation associated with the Fbn2 locus was engineered into the equivalent locus in mouse to generate potential models for the disease. This mutant line was created through the CRISPR/Cas9 technology, so that the desired mutation was directly introduced into the mouse genome in oocytes, with no ES cell manipulation necessary. Another disease model that was successfully accomplished this year with a similar approach is the engineering of the Cyp4v3 knockout allele for Bietti Crystalline Dystrophy. 2) Reverse genetic confirmation of disease candidate genes: The core supports disease gene discovery programs by making knockouts and knockins of candidate genes to establish direct linkage between the genes identified in forward genetic screens and the genetic conditions against which the screens were conducted. Projects in this area during the past year include generation of knockin and knockout mutations for RPE65, Rd9 and Asph1 loci. 3) Functional genomic studies of genes with interesting expression patterns and predicted to be functionally important in physiology and pathology: Most of the current gene targeting projects are aimed at simply understanding the functions of various genes relevant to NEI as well as other participating IC research programs. Work in this area currently include the characterization of the miR183 cluster knockout, which is involved in sensory neuron function and deletion of which has displayed phenotypes of retinal dysfunctions; completion of the construction of the conditional knockouts of the miR204 and miR211 genes, which have shown in preliminary studies involved in maintenance of RPE normal functions; and generating inducible expression of a beta-actin-egfp fusion allele in the inner ear. During the past year, we have: * worked on 48 different gene targeting projects of total 15 distinct loci at various stages * made 78 different constructs for gene targeting in ES cells and CRISPR/Cas9-meduated gene targeting in mouse zygotes, or for over-expression of gene of interest in ES cells or zygotes * made 70 endotoxin-free, large scale DNA preparations of DNA constructs * conducted 8 electroporation experiments plus 8 transfection assays for targeted mutagenesis in ES cells * picked, expanded and cryopreserved 2000 individual ES clones * Expanded 130 positive ES clones * Assisted in PCR screening of targeted clones by performing genomic PCR (2350 reactions) * derived 2 ES lines from one transgenic mouse line carrying a reporter allele * assisted in iPS cell culture experiments for total of 4 lines derived last year at GEC * transfected 2 lines of immortalized MEF cells immortalized at GEC last year * injected 12 ES cell lines into mouse embryos to generate 35 chimeric mice * Injected 2 DNA constructs into fertilized mouse oocytes to generate 1 transgenic mouse * injected CRISPR/Cas9 constructs into fertilized mouse oocytes for 58 injection sessions to produce 57 mutant mice * isolated DNA from 10,727 mouse tail biopsy samples * performed 8,708 PCR reactions to genotype mice in the facility * set up 2,955 matings to propagate mouse lines * completed or oversaw weaning, tagging, and tail biopsy of 10,944 mice born in the facility * rederived 6 mouse lines * worked on cryopreservation of 137 mouse lines freezing 4,977 mouse embryos at the two cell stage, and 940 straws of sperm. * cryopreserved 4 lines of zebrafish as frozen sperm and validated 4 lines. Cryopreserved testes from one line of zebrafish. * performed assisted reproduction to save 26 mouse lines from extinction and/or reconstitute mouse lines from frozen stock. * worked on several special projects which do not fall neatly into the above categories. These services and collaborative services were performed for 17 PIs from 5 NEI labs (LI, LRCMB, N-NRL, OGVFB, OSD), plus 2 PIs from 1 other institute at NIH (NIDCD)